Intramolecular Fluorescence Resonance Energy Transferbetween Fused Autofluorescent Proteins RevealsRearrangements of the N- and C-terminal Segments of thePlasma Membrane Ca2� Pump Involved in the Activation*

Received for publication, April 23, 2007, and in revised form, September 26, 2007 Published, JBC Papers in Press, September 27, 2007, DOI 10.1074/jbc.M703377200

Gerardo R. Corradi and Hugo P. Adamo1

From the IQUIFIB, Facultad de Farmacia y Bioquımica, Universidad de Buenos Aires, Junın 956, Buenos Aires 1113, Argentina

The blue and green fluorescent proteins (BFP and GFP)have been fused at the N- and C-terminal ends, respectively,of the plasma membrane Ca2� pump (PMCA) isoform 4xb(hPMCA4xb). The fusion protein was successfully expressed inyeast and purified by calmodulin affinity chromatography.Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme withrespect to Ca2�-ATPase activity and sensitivity to calmodulinactivation. In the autoinhibited state BFP-PMCA-GFP exhib-ited a significant intramolecular fluorescence resonance energytransfer (FRET) consistent with the location of the fluorophoresat an average distance of 45 A. The FRET intensity in BFP-PMCA-GFP decreasedwhen the enzymewas activated either byCa2�-calmodulin, partial proteolysis, or acidic lipids. More-over, FRET decreased and became insensitive to calmodulinwhen hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of thePMCA are in close proximity in the autoinhibited conforma-tion, and they separate or reorient when the PMCA achievesits final activated conformation.

The homeostasis of the intracellular Ca2� is crucial for cellfunction. The Ca2� ATPases from plasma membrane(PMCAs)2 participate in themodulation ofCa2� signals and areresponsible for the long term maintenance of the low concen-tration of intracellular Ca2� (1). The PMCAs belong to theP2-type ATPase superfamily of ion pumps and form an aspartylphosphate intermediate during the transport cycle (2). Anotheressential feature of these ATPases is their ability to switch

between two different conformational states from E2 to E1 inthe presence of the transported ion.Human PMCAs are encoded by four separate genes, and

additional variants are generated via alternative splicing of pri-mary gene transcripts. PMCA4 is found virtually in all humantissues, and the splice variant xb is the most studied isoform.Computer modeling and sequence comparisons indicate thatthe overall structure of the PMCAs closely resembles that ofother P-ATPases. Following the domain organization proposedfor the SERCA (3, 4), the PMCA would contain a transmem-brane region of 10 segments (M1–M10) and three major cata-lytic domains exposed to the cytosol. The nucleotide-binding(N) and the phosphorylation (P) domains contain the ATPbinding site and the aspartate residue that forms the acyl phos-phate intermediate, respectively, whereas the actuator domain(A) plays an essential role in the long range transmission of theconformational changes occurring during the transport cycle.Despite the clear overall homology, certain amino acid seg-

ments of the PMCAprotein are not found in SERCA. The exist-ence of these segments in the PMCAmolecule has been gener-ally associated with the extensive regulatory mechanisms thatare known to alter the function of the PMCA. Indeed, themajordifference between the two calcium pumps is the long C-termi-nal segment (C region) of the PMCA following M10, and thisregion is involved in the so-called intrasteric inhibition of thepump (5). This type of control of enzymatic activity by inhibi-tory sequences acting directly at the active site of the enzyme ischaracteristic of protein kinases and phosphatases in which theautoinhibitory sequences resemble their substrates (6). In anal-ogy with activationmechanism of these enzymes, it is proposedthat in the resting state the autoinhibitor interacts with thecatalytic region maintaining the PMCA inhibited. Calmodulinis the main activator of the pump, but a variety of mechanisms,including proteolysis, binding of acidic lipids, and phosphoryl-ation by protein kinases, have been shown to switch partially ortotally the activity of the PMCA back on. In the hPMCA4xbisoform the C-terminal autoinhibitor region has been found tocontain two domains. A sequence of 28 amino acids identifiedas domain “C” (residues 1086–1113) binds calmodulin andacidic lipids and is responsible for �80% of the inhibition. Thebinding of calmodulin to domain C enhances both the Ca2�

sensitivity and the turnover of the pump. Alternative splicing atthe so-called alternative spliceCmodifies the calmodulin-bind-ing autoinhibitory region leading to isoforms with different

* This work was supported in part by the Universidad de Buenos Aires (GrantB029), by the Consejo Nacional de Investigaciones Cientıficas y Tecnicas(CONICET) (Grant PIP 6168), and by ANPCyT BID 1728 OC-AR PICT15-25965. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 An established investigator of CONICET. To whom correspondence shouldbe addressed. Tel.: 5411-4964-8289 (ext. 125); Fax: 5411-4962-5457; E-mail:hpadamo@qb.ffyb.uba.ar.

2 The abbreviations used are: PMCA, plasma membrane Ca2� pump; h4xb,plasma membrane Ca2� pump derived from the human gene 4 (spliced formx at the splicing site A and spliced form b at the splicing site C); FRET, fluores-cence resonance energy transfer; GFP, green fluorescent protein; BFP, bluefluorescent protein; PC, phosphatidylcholine; SERCA, sarcoplasmic-endoplas-mic reticulum calcium ATPase; C12E10, polyoxyethylene-10-laurylether.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 49, pp. 35440 –35448, December 7, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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responsiveness toward calmodulin activation (7). Full inhibi-tion has been found to require a segment further downstreamthe calmodulin binding site (8). This second inhibitorysequence, which has been called domain “I,” encompass resi-dues 1114–1157. Phosphorylation by protein kinase C ofdomain I residues has been shown to result in a partial activa-tion of the pump in the absence of calmodulin (9).Cross-linking experiments using a synthetic peptide C28W

made after the sequence of the autoinhibitory domain C ofhPMCA4xb revealed that the N-terminal half of this sequenceinteracts with the N domain (Cys537–Thr544), whereas theC-terminal part interacts with the A domain (Ile206–Val271) ofthe catalytic core (10, 11). It has been hypothesized that thebinding of calmodulin to the C28W sequence in the pumpinduces a conformational change in the autoinhibitory domainthat disrupts its interaction with the catalytic domain, therebyactivating the pump. Consistently with this model it has beenshown that the C-terminal segment of the hPMCA4xb is moreaccessible to proteases when the pump in an activated openstate than in the inhibited closed state (12). Furthermore, theactivating effect of mutations of Asp1080 has been suggested toarise from the impaired function of the region preceding theautoinhibitory sequence as a hinge between the regulatory andcatalytic regions (13).Although the concept that activation of the PMCA involves

conformational changes associated with the removal of theautoinhibitory domain from the active site is widely acceptedthere have been no directmeasurements of the rearrangementsthat take place in the PMCA during the activation. Moreover,the degree in which the C-terminal sequence downstreamdomain C participates in the conformational changes associ-ated, which would occur during activation, is not clear at thistime. In this study we have explored the possibility of obtaininga functional PMCA labeled with two autofluorescent proteinsand its potential use as a reporter of conformational changesassociated with the activity of the enzyme.

MATERIALS AND METHODS

Chemicals—Polyoxyethylene-10-laurylether (C12E10), L-�-phosphatidylcholine Type X-E (P-5394) from dried egg yolk,phosphodiesterase 3�,5�-cyclic nucleotide activator (calmodu-lin) from bovine brain, calmodulin-agarose, calcimycin(A23187), trypsin, ATP (disodium salt, vanadium-free), SDS,yeast synthetic drop-out media supplement without leucine,yeast nitrogen base without amino acids, dextrose, enzymesand cofactors for the synthesis of [�-32P]ATP, and all otherchemicals were obtained from Sigma. Tryptone and yeastextract were fromDifco. Carrier-free [32P]H3PO4was providedby PerkinElmer Life Sciences (Boston, MA). [�-32P]ATP wasprepared by themethod of Glynn andChappell (14) except thatno unlabeled orthophosphate was added to the incubationmedium. Salts and reagents were of analytical reagent grade.Constructions of the DNA Coding for BFP-PMCA-GFP—The

cDNA coding for PMCA-GFPwas obtained by a two-step PCR in astandardmegaprimer protocol (15) using PfuDNA polymerase andprimers A (5�-GCCCATGGCCTGTGATGGACT-3�), B (5�-GTC-AAAGCGTTCCATAGTTCCGGAGGTGGAGCTAGCAAAGG-3�), and C (5�-TATTTAGGGCCCTCAATCGATGTTGTACAG-

TTCATCCA-3�). Primer C contains a restriction site for nucleaseApaI at its 3� position, whereas primer A anneals to the hPMCA4xbDNAupstream of a naturally occurring BspEI unique site. Dur-ing the first PCR a fragment was amplified using the Super-GloGFP cDNA (Qbiogene) as template and primers B and C.The 750-bp B-C productwas isolated by electrophoresis in a 1%agarose gel and extracted using DNA QIAEX II (Qiagen). Thepurified BC fragment was used in the next PCR step along withprimer A using the cDNA coding for hPMCA4xb as template.The 2144-bp A-BC fragment was digested with BspEI and ApaIand subcloned into the corresponding position of hPMCA4xbinserted into the XhoI-ApaI sites of the pMP625 vector (16).To obtain the cDNA coding for BFP-PMCA first a unique

site for nucleaseMluI was introduced at position 4 of the cDNAof hPMCA4xb. To this end a DNA fragment was amplified byPCR using oligonucleotides CA2 (5�-TGCAGGTCGACCAT-GACGCGTCCATCAGACCGTGT-3�) and 308 (5�-TCTCCA-GGATGATGAGCGTGACAT-3�) digested with SalI andDraIIIand cloned back at the corresponding position of hPMCA4xb. ADNA fragment was amplified by PCR using the SuperGlo BFPcDNA (Qbiogene) as template and the pair of primers,5�-BGMLUI and 3�-BGMLUI. Primer sequences were as fol-lows: 5�-BGMLUI, 5�-ATAATAACGCGTGGAGGTGGAGC-TAGCAAAGGAGAAGAACTCTTC-3�; and 3�-BGMLUI, 5�-AATTATACGCGTTCCGCCTCCATCGATGTTGTACAG-TTCATCCA-3�. Primers 5�-BGMLUI and 3�-BGMLUIincluded a restriction site for nuclease MluI. The product5�-BGMLUI/3�-BGMLUI was isolated by electrophoresis in a1% agarose gel and extracted using DNA QIAEX II (Qiagen).The isolated fragment was digested with MluI and subclonedinto the hPMCA4xb cDNA containing aMluI site at position 4.The DNA coding formutant BFP-PMCA-GFPwas obtained byreplacing the wild-type XhoI-BspEI fragment of PMCA-GFPwith the SalI and BspEI fragment of BFP-PMCA.Yeast Strain, Transformation, and Growth Media—Saccha-

romyces cerevisiae strain DBY 2062 (MATa his4–619 leu2–3,112) (17) was used for expression. Yeast cells were trans-formed with the pMP625 vector containing a Leu� marker andthe PMAI promoter. For transformation with the plasmid con-struct a lithium acetate/polyethylene glycol method was uti-lized (18). The cells were grown in completemedia (0.75% yeastextract, 1.13% Tryptone, 2.2% dextrose), and transformantswere selected for their ability to grow in the absence of leucineon plates containing 6.7% yeast-nitrogen base without aminoacids (YNB), 0.67% complete supplemented medium minusLeu (Leu�), 2.2% dextrose, and 1.5% agar.Membrane Isolation and Purification of Recombinant

BFP-PMCA-GFP—Four liters of yeasts expressing the BFP-PMCA-GFPproteinwere grown inYNBLeu�media at 28 °C inglass flasks with agitation, and, after the culture reached anapproximate A600 of 1.5, 4 liters of complete media was added,and the incubation continued for 6 h (A600 � 4.0–5.0). Totalyeast membranes were solubilized with C12E10, and the PMCAwas purified by calmodulin-affinity chromatography asdescribed previously (19). �400 �g of purified PMCA wasobtained from one batch of yeast. For the calculation of themolar concentration values the Mr of BFP-PMCA-GFP was

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taken as 190,000 and that of the single labeled PMCAs as162,000.Protein Assay—The protein concentration was initially esti-

mated by themethod of Bradford (20) using bovine serumalbu-min as a standard. To achieve a better assessment of the contentof PMCA protein in each preparation, the samples were ana-lyzed by SDS-PAGE using PMCA purified from porcine redcells as standard, and the intensity of the bands was comparedafter staining the gels with Coomassie Blue.Reconstitution of BFP-PMCA-GFP in Proteoliposomes—Pro-

teoliposomes containing the BFP-PMCA-GFP protein wereobtained as described by Hao et al. (21) with a slight modifica-tion. Briefly, 1 ml of purified BFP-PMCA-GFP (�100 �g ofprotein) was added to 250 �l of a previously sonicated mixtureof 29 mg/ml phosphatidylcholine and 57 mg/ml C12E10. Thedetergent/protein/phospholipid mixture was kept for 5 minunder gentle stirring, and then the detergent was partiallyremoved by adding 0.25 g of prewashed wet SM-2 Bio-Beads(Bio-Rad). The mixture was stirred at room temperature for 30min, and the Bio-Beads were separated by filtration.Ca2�-ATPase Activity—The Ca2�-ATPase activity was esti-

mated from the release of [32P]Pi from [�-32P]ATP at 37 °C (22)in 0.3 ml of ATPase reaction medium containing 50 mMHEPES-K (pH 7.00 at 37 °C), 100mMKCl, 4mMMgCl2, 500�MEGTA, 2 �M A23187, 3 mM [�-32P]ATP and enough CaCl2 andcalmodulin to give the Ca2� and calmodulin concentrationsindicated in each experiment. The reaction was initiated by theaddition of 2 �g of BFP-PMCA-GFP reconstituted in proteoli-posomes andwas terminated by acid denaturation after 30min.During this period the amount of Pi liberated from ATPincreased linearly with time. The free Ca2� concentration inreaction medium was calculated using the Fabiato and Fabiatoprogram (23) using the Blinks constants for EGTA/Ca2�,EGTA/Mg2�, ATP/Ca2�, and ATP/Mg2�.Ca2� Uptake—For measuring the Ca2� uptake activity 272

�g of BFP-PMCA-GFP was reconstituted in proteoliposomesas described above except for that the reconstitution mediacontained 5 mM of potassium oxalate. The detergent wasremoved by incubating the protein with 0.19 g of prewashedwet SM-2 Bio-Beads. The mixture was stirred at room temper-ature for 30 min, then 0.19 g more of prewashed wet SM-2Bio-Beads was added, and the incubation was continued for 30min. The Bio-Beads were separated by filtration, the samplewas diluted with 3 ml of 160 mM KCl, 20 mM Tris-HCl (pH 7.4at 25 °C), and the proteoliposomes were collected by centrifu-gation at 40,000� g for 30min. TheCa2� uptakewasmeasuredas described previously (24). The reaction mixture contained100mMKCl, 50mMTris-HCl (pH 7.4 at 37 °C), 1.5mMATP, 95�MEGTA, 2.5mMMgCl2, andCaCl2 (labeled with 45Ca) to give10 �M free Ca2�. The proteoliposomes (40 �l) were incubatedin the reaction mixture at 25 °C for 5 min, and the reaction wasinitiated by the addition of ATP. The reaction was finished byfiltering the samples through a 0.2-�m filter. The 45Ca taken upby the liposomes was determined by counting in a scintillationcounter. The radioactivity retained in the filter in the absence ofATP was subtracted from each data point.Western Blotting and Protein Staining—SDS electrophoresis

and immunoblotting were carried out as previously described

(25). Proteins were electrophoresed on a 7.5% acrylamide gelaccording to Laemmli (26) and revealed by Coomassie Bluestaining, or subsequently electrotransferred onto MilliporeImmobilon P membranes. Nonspecific binding was blocked byincubating themembranes overnight at 4 °C in a solution of 160mMNaCl, 0.05%Tween 20, and 1% non-fat drymilk. Themem-branes were incubated for 1 h with 5F10 antibody (27, 28) fromascitic fluid (dilution 1:1000). For staining, biotinylated anti-mouse immunoglobulin G and avidin-horseradish peroxidaseconjugate were used.Tryptic Digestion of BFP-PMCA-GFP—Digestion of BFP-

PMCA-GFP with trypsin was done at 25 °C, in a medium con-taining 5 �g/ml of BFP-PMCA-GFP, 0.25 mg/ml C12E10, 0.12�g/ml PC, 100 mM KCl, 50 mM Tris-HCl (pH 7.25 at 25 °C), 5mM MgCl2, and 500 �M EGTA. The reaction was initiated bythe rapid addition of 0.5 �g/ml trypsin, and the emission spec-tra were registered at different times as indicated in the eachfigure.For SDS-PAGE 19 �g of BFP-PMCA-GFP was suspended in

3.6 ml of the same media as above, and the reaction was initi-ated by the addition of 2 �g of trypsin. At different times 200 �lof the reaction was removed and mixed with 20 �l of 100%trichloroacetic acid. The proteinwas precipitated and dissolvedin sample buffer, and the proteolytic products were separatedby electrophoresis in 9% acrylamide gel. The gel was stainedwithCoomassie Blue and scanned for fluorescence at 520 nm ina Storm 840 scanner.Fluorescence Measurements—The fluorescence measure-

ments were performed in an SLM-Aminco-Bowman spec-trofluorometer (Spectronic Instrument Inc., Rochester, NY)using a 3- � 3-mm quartz cuvette. The media contained 5�g/ml PMCA-fused fluorescent protein, 0.25 �g/ml C12E10,0.12�g/ml PC, 100mMKCl, 50mMTris-HCl (pH7.25 at 25 °C),5 mM MgCl2, and 500 �M EGTA. The final volume was 150 �l.The temperature was set at 25 °C. The excitation and emissionbandwidths were set at 4 nm. Appropriate blanks correspond-ing to the reaction media were subtracted from the measure-ments to correct for background fluorescence.

FIGURE 1. The fusion protein BFP-PMCA-GFP is correctly expressed in S.cerevisiae. The wild-type PMCA and the PMCA containing the fusedautofluorescent proteins were purified from yeast membranes and submit-ted to SDS-PAGE in a 7% gel. Proteins were stained with Coomassie Blue(lanes 1–5) or transferred to polyvinylidene difluoride membranes, and thePMCA was detected with the monoclonal antibody 5F10 (lanes 6 and 7). Lane1, 0.45 �g of PMCA purified from pig erythrocytes; lane 2, 0.45 �g of recom-binant wild-type PMCA; lane 3, 0.50 �g of BFP-PMCA; lane 4, 0.50 �g of BFP-PMCA; lane 5, 0.70 �g of BFP-PMCA-GFP; lane 6, 0.1 �g of recombinant wild-type PMCA; and lane 7, 0.1 �g of BFP-PMCA-GFP.

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The efficiency of energy transfer (E) was estimated as thefraction of donor molecules de-excited via energy transfer tothe acceptor, E � (1 � Ida/Id), where Ida and Id are the intensityof emission of the donor (BFP) in the presence and in theabsence of acceptor, respectively. To achieve a more precisedetermination of the values of Ida and Id the integral of the areaunder the curve between 430 nm and 470 nm was taken. Theestimation of the distance between the fluorophores was doneaccording to the Forster equation, E � 1/(1� rda6/R06), whererda is the distance between the donor (BFP) and acceptor (GFP)and R0 (Forster distance) is the distance at which 50% theenergy is transferred. R0 depends on the quantum yield of thedonor (�d), the extinction coefficient of the acceptor (�d),the overlap of the donor emission and acceptor excitationspectra (Jda), and the mutual orientation of the chro-mophores (�2) (29). A Forster distance (R0) of 40 Å was usedfor the fluorophore pair BFP-GFP (30, 31), and a randomorientation (�2 � 2/3) was assumed for distance calculations.

RESULTS

We have previously found that the extreme N-terminal seg-ment of the hPMCA4xb (residues 1–6) is not critical for theactivity of the PMCA (32). Accordingly, the BFP was fused atthe N-terminal segment of after Thr2. The GFP was fused afterthe last C-terminal residue at�100 residues downstreamof thecalmodulin binding-autoinhibitory sequence. A Gly-Gly-Glyspacer was introduced between the PMCA and the fluorescentproteins to increase the chance that the fluorophores wouldexhibit a random orientation as assumed for distance calcula-tions from the FRET efficiency.Total membranes from yeast cells transformed with the

expression vector pMP625 containing the DNA coding for theautofluorescent PMCA fusions were isolated and solubilized bythe detergent C12E10, and the dual- or single-labeled PMCAproteins were purified by calmodulin-affinity chromatographyfollowing the same protocol used for the purification of thewild-type PMCA. As shown in Fig. 1 the SDS-PAGE of thepurified proteins revealed the presence of one major polypep-tide, which migrated according to the expected size.Results in Fig. 2A show that BFP-PMCA-GFP was capable of

hydrolyzing ATP. The Ca2�-ATPase activity of BFP-PMCA-GFP increasedwith increasing concentrations ofCa2� reachinga maximal activity of 0.4 �mol/mg/min at �10 �M Ca2�. Theaddition of 200 nM calmodulin increased the maximal activityof BFP-PMCA-GFP and reduced the concentration of Ca2�

needed for half-maximal activity from 0.5 to 0.1 �M Ca2�. Asshown in Fig. 2B, half-maximal activation of BFP-PMCA-GFPwas attained at 5 nM calmodulin. In the presence of 1 �M Ca2�

the activity of the BFP-PMCA-GFP enzyme in the absence ofcalmodulin was �40% of that attained at saturating concentra-tions of calmodulin. This value of basal activity is similar to thatof the recombinant wild-type hPMCA4xb enzyme obtainedfrom yeasts under similar conditions (19). These results suggestthat the inhibitory function of the C-terminal regulatory

FIGURE 2. BFP-PMCA-GFP is a functional calmodulin-regulated Ca2�-ATPase. A, Ca2�-ATPase activity of BFP-PMCA-GFP as a function of the Ca2�

concentration in the presence of 0 (E) or 200 nM (F) calmodulin. The maximalactivity in the presence of calmodulin was taken as 100%. The lines representthe best fit to the data given by the Hill equation with the following parame-ters: in the absence of calmodulin, Vmax � 65 � 3%, K0.5 � 0.50 � 0.07 �M,nH � 1.2 � 0.2; in the presence of calmodulin, Vmax � 96 � 3%, K0.5 � 0.14 �0.01 �M, nH � 3.5 � 1.4. B, calmodulin dependence of BFP-PMCA-GFP. TheCa2� ATPase activity of the BFP-PMCA-GFP protein was measured at increas-ing concentrations of calmodulin in presence of 1 �M Ca2�. The line repre-sents the best fit to the data given by the equation, V � V0 � Va/(1 � Kcam/calmodulin), with the following parameters, V0 � 38 � 6%, Kcam � 5 � 2 nM,Va � 63 � 7%. C, Ca2� uptake by proteoliposomes containing BFP-PMCA-GFP. The preparation of proteoliposomes and the measurement of 45Ca2�

uptake are described under “Materials and Methods.” The concentration ofCa2� was 10 �M. The average (�S.D.) cpm of 45Ca2� taken up per 40 �l of

proteoliposome suspension of an experiment conducted in triplicate isshown. The continuous line represents the best fit given by the equation, v �V(1 � e�kt), with V � 983 cpm/40 �l and k � 0.015 s�1.

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domain was preserved despite the addition of the GFP. In addi-tion results in Fig. 2C show that after reconstitution in proteo-liposomes BFP-PMCA-GFP supported the ATP-dependentuptake of Ca2�, further reinforcing the idea of the functionalcompetence of the PMCA fusion protein.The BFP-PMCA-GFP protein exhibited maximum excita-

tion and emission peaks at near the same wavelength that theisolated fluorescent proteins. Fig. 3A shows the fluorescenceemission spectra of BFP-PMCA and BFP-PMCA-GFP whenthe BFP fluorophore was excited at 387 nm and that of PMCA-GFP excited at 474 nm. The fluorescence emission of BFP-PMCA-GFP was notably lower than that of BFP-PMCA at theBFP emission wavelength, and the fluorescence intensityincreased in the region of the spectra corresponding to the GFPemission. This was indicative of the existence of energy transferbetween the two fluorescent proteins.Previous studies have shown that the purified hPMCA solu-

bilized with detergents undergoes a reversible self-association(33–35). To investigate if association between BFP-PMCA-GFP molecules was causing FRET, the emission of 18 nM BFP-PMCA was titrated with increasing amounts of PMCA-GFPeither in the presence of 0.5 mM EGTA or 1 �M Ca2�. Fig. 3Bshows that in the absence of Ca2� the emission spectra of mix-tures of BFP-PMCA and PMCA-GFP up to 30 nM were similarto that of the BFP-PMCA alone. At higher concentrations ofPMCA-GFP the relative fluorescence at 509 nm increased sug-gesting that at these concentrations energy was transferredbetween BFP-PMCA and PMCA-GFP. In the presence of Ca2�

(Fig. 3C) the enhancement of the PMCA-GFP fluorescence waseven more evident in agreement with the idea that the oli-gomerization of the enzyme is favored by Ca2� (36).

The results shown in Fig. 3 suggest that, at the concentrationof BFP-PMCA-GFP used in Fig. 3, FRET arises from the inter-action between fluorophores belonging to the same molecule.The efficiency transfer estimated by comparing the emission ofBFP-PMCA-GFP and BFP-PMCA at 450 nM was 52%. If a freeorientation of donor-acceptor pair is assumed, the calculatedaverage distance betweenBFP andGFP in the BFP-PMCA-GFPprotein was 45 Å.It has been previously shown that a limited exposure of the

PMCA to trypsin leads primarily to the removal of peptidesfrom both ends of the molecule (37). Fig. 4A shows the effect ofthe treatment with trypsin on the BFP-PMCA-GFP emissionspectra. As the time of proteolysis increased, the relative emis-sion at 509 nm decreased, consistently with the idea that thecleavage of the fluorescent proteins from the PMCA increasedthe separation between them. Interestingly the decline of FRETwith the time of proteolysis was biphasic (Fig. 4B) with a fastcomponent accounting for �25% of the total and a larger com-ponent decaying much slower. Proteolysis of BFP-PMCA-GFPwas also followed by SDS-PAGE. Fig. 4C shows the productionof proteolytic fragments revealed by Coomassie Blue staining,

FIGURE 3. BFP-PMCA-GFP exhibits intramolecular FRET. A, comparisonbetween the emission spectra of BFP-PMCA, PMCA-GFP, and BFP-PMCA-GFP.Each fusion protein was adjusted at a concentration of 26 nM in a standardmedia as described under “Materials and Methods.” The excitation wave-length was 387 nm for BFP-PMCA and BFP-PMCA-GFP and 474 nm for thePMCA-GFP. B, titration of BFP-PMCA emission by PMCA-GFP in the absence ofCa2�. 18 nM BFP-PMCA was added to the standard media containing 0.5 mM

EGTA and without added CaCl2. The BFP-PMCA was excited at 387 nm, andthe emission spectrum was recorded (thinner line). Then, increasingamounts of PMCA-GFP were added rising the total concentration of singlelabeled PMCA to 24, 29, 35, and 40 nM (progressively thicker line). After eachaddition the mixture was allowed to equilibrate for 5 min before the emis-sion spectra were recorded. Appropriate blanks corresponding to thereaction media plus the corresponding amount of PMCA-GFP were

subtracted from the measurements to correct for background fluorescence,and the fluorescence intensities were normalized to the maximum emissionof the BFP fluorophore. C, titration of BFP-PMCA emission by PMCA-GFP in thepresence of Ca2�. The experiment was conducted as in B, except that CaCl2was added to the media to give 10 �M Ca2�.

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whereas Fig. 4D shows the scan of the same gel for GFP fluo-rescence. As the time of proteolysis increased the BFP-PMCA-GFP was digested to a peptide of apparent molecular mass of170 kDa and then to other peptides of lower apparentmolecularmass. Because the 170-kDa peptide did not exhibit GFP fluo-rescence the conversion of BFP-PMCA-GFP to the 170-kDapeptide must have involved the cleavage of the GFP moiety. Atthe longest digestion times all the detected fluorescence was ina peptide of apparent molecular mass�35 kDa probably corre-sponding mostly to the free GFP. The time course of theappearance of the 170-kDa fragment is in good agreement withthe fast phase of FRET decay. Interestingly, the amount of 170-kDa fragment began to decrease after 2 min of proteolysis fol-lowing a time course similar to the slow phase of FRET decay.Fig. 5A shows the effect of Ca2�-calmodulin on the fluo-

rescence emission of BFP-PMCA-GFP. In the presence of 10�M Ca2� the emission spectra were not significantly differ-ent from those observed in the absence of Ca2�. However,the addition of 10 �M Ca2� plus 200 nM calmodulindecreased the intensity of fluorescence attributable to FRET.This effect was almost fully reversed by the removal of Ca2�

with excess of EGTA. Fig. 5B shows that the fluorescenceintensity of the PMCA-GFP did not change when it was sub-mitted to identical experimental conditions.

The dependence of the intensityof FRET with the concentration ofcalmodulin was examined in moredetail in Fig. 6. The decrease ofFRET produced by increasing con-centrations of calmodulin occurredin parallel with the activation of theCa2�-ATPase. At saturating con-centrations of Ca2�-calmodulin thecalculated average distance betweenthe fluorophores in the BFP-PMCA-GFP increased to 50 Å.It is well known that acidic lipids

can mimic the activation of thePMCAproduced by calmodulin (38,39). As shown in Fig. 7, when BFP-PMCA-GFP was activated by phos-phatidylinositol the intensity ofFRETwas similar to that attained bythe enzyme in the presence of PCplus Ca2�-calmodulin. Moreover,in the presence of phosphatidylino-sitol the further addition of Ca2�-calmodulin produced only a minoreffect on the emission spectra ofBFP-PMCA-GFP.To attain a conformationwith the

maximal catalytic activity thePMCA is likely to undergo sequen-tial conformational rearrange-ments. Thus, the most importantpoint of interest was to investigatewhich step of the BFP-PMCA-GFPactivationwas being reported by the

decrease in FRET. For this purpose we took advantage of theactivating effect of mutation D170N reported previously (19).As shown in Fig. 8 in the absence of Ca2� FRETwas lower in themutant BFP-PMCA(D170N)GFP than in the wild-type BFP-PMCA-GFP, accounting for an efficiency transfer of �26%.Neither the addition of Ca2� nor Ca2�-calmodulin produced asignificant change in the BFP-PMCA(D170N)GFP spectra.

DISCUSSION

FRET is a highly sensitive method used for investigating thestructure and dynamics of macromolecules (40). Frequently,however, the attachment of the required fluorophores is detri-mental to protein function. One of the questions posed at abeginning of this work was whether a double-labeled BFP-PMCA-GFP containing two large fluorescent protein moietiescould be generated without significantly disturbing the func-tion on the enzyme. The results reported here show that, byfusing the fluorescent proteins at both ends of the PMCA, wehave succeeded in creating such a construct. We found thatBFP-PMCA-GFPpossessesCa2�-dependentATPase andATP-dependent Ca2�-pumping activities. Moreover, the Ca2�-ATPase activity of BFP-PMCA-GFP in the absence of calmod-ulin and its apparent affinity for calmodulin were similar to thevalues reported for the wild-type enzyme (19), suggesting that

FIGURE 4. Proteolysis of BFP-PMCA-GFP. A, effect of proteolysis of BFP-PMCA-GFP on its emission spectra.The BFP-PMCA-GFP was submitted to proteolysis with trypsin as described under “Materials and Methods.” Theemission spectrum was taken before adding the protease (uppermost thicker curve) and again at 2, 4, 10, 15, 20,25, 30, 35, and 40 min (progressively thinner curves) after the proteolysis was started by the addition of theprotease. The excitation wavelength was 387 nm. The fluorescence intensities were normalized to the maxi-mum emission of the BFP fluorophore. B, FRET estimated as the average fluorescence intensity of GFP between500 and 510 nm for each curve in A and corrected by the background BFP fluorescence was plotted as afunction of the time of proteolysis. The fluorescence intensity without added protease was taken as 100%. Theline is the best fit to the data given by a double exponential equation using the following parameters: Af � 24 �4%, kf � 1.2 � 0.8 min�1, As � 76 � 3%, and ks � 0.024 � 0.001 min�1, where Af and As represent the maximumamplitude of the fast and slow phase, respectively. C, SDS-PAGE analysis of the proteolytic digestion of BFP-PMCA-GFP. The BFP-PMCA-GFP protein was submitted to proteolysis with trypsin as described under “Mate-rials and Methods” under similar conditions as those in A for the time (minutes) indicated at the top of each lane.2 �g of total protein was loaded in each lane and electrophoresed in 9% acrylamide gel. M, molecular massmarker. The peptides were visualized by Coomassie Blue staining. D, GFP fluorescence of proteolytic fragmentsof BFP-PMCA-GFP. The same gel shown in C was scanned for fluorescence emission at 520 nm.

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the fused proteins did not impede the autoinhibition of theenzyme.The second concern in this study was whether the fluoro-

phores would get close enough in the BFP-PMCA-GFP mole-cule to display FRET. This was indeed the case, because in theabsence of Ca2�-calmodulin and acidic lipids we observed sig-nificant FRET in BFP-PMCA-GFP. The PMCA purified fromhuman red cells has been previously observed to form dimers,and the reported concentration of PMCAneeded for half-max-imal dimerization varies between 17 and 370 nM (33–35). Thus,FRET in BFP-PMCA-GFP could reflect both the interaction offluorophores from the same molecule and from associatedPMCA molecules. However, at a concentration of proteinbelow 30 nM, we did not detect significant FRET when the flu-orescent proteins were attached to different PMCAmolecules.

Thus, at low protein concentration the FRET we observed inBFP-PMCA-GFP was due to the interaction of fluorophoresfrom the same molecule.The values of energy transfer have been frequently translated

as distance between the probes (40). The value of FRETobserved in the BFP-PMCA-GFP protein indicates that bothprobes would be an average of �45 Å apart. Thus, the BFP andGFP would be indeed very close in the BFP-PMCA-GFP,because both fluorophores cannot come closer than 30 Å fromone another as they are each buried �15 Å within the fluores-cent protein. It should be noted that the calculated distance hasto be takenwith caution, because it is the average of the distance

FIGURE 5. A, effect of Ca2�-calmodulin on the emission spectra of BFP-PMCA-GFP. The concentration of BFP-PMCA-GFP was adjusted at 26 nM in a standardmedia as described under “Materials and Methods.” The sample was excitedat a wavelength of 387 nm, and the fluorescence emission spectra wererecorded (solid line). The spectra were taken again after the sequential addi-tion enough CaCl2 to give 10 �M free Ca2� (long dashed line), 200 nM calmod-ulin (short dashed line), and excess of 500 �M EGTA (dotted line). The fluores-cence intensities were normalized to the maximum emission of the BFPfluorophore. B, effect of Ca2�-calmodulin on the emission spectra of PMCA-GFP. The concentration of PMCA-GFP was adjusted at 30 nM in a standardmedia as described under “Materials and Methods.” The sample was excitedat a wavelength of 474 nm, and the fluorescence emission spectra wererecorded (solid line). The spectra were taken again after the sequential addi-tion enough CaCl2 to give 10 �M free Ca2� (long dashed line), 200 nM calmod-ulin (short dashed line), and excess of 500 �M EGTA (dotted line). The fluores-cence intensities were normalized to the maximum emission of the GFPfluorophore.

FIGURE 6. Dependence of the energy transfer (E) and the activation of theBFP-PMCA-GFP enzyme on calmodulin concentration. The efficiency ofenergy transfer (E) in BFP-PMCA-GFP was estimated from the ratio betweenthe fluorescence intensity of BFP-PMCA-GFP and BFP-PMCA at increasingconcentrations of calmodulin as described under “Materials and Methods.”The value of E in the absence of calmodulin minus that at 200 nM calmodulinwas taken as 100%. The activation of BFP-PMCA-GFP was estimated from theCa2�-ATPase activity at each concentration of calmodulin minus the activityin the absence of calmodulin. The activity at 200 nM calmodulin minus that inthe absence of calmodulin was taken as 100%.

FIGURE 7. Effect of acidic lipids on the emission spectra of BFP-PMCA-GFP. The concentration of BFP-PMCA-GFP protein was adjusted to 18 nM in astandard media containing either 0.12 �g/ml PC (thin solid line); 12 �g/ml PC,10 �M Ca2�, and 200 nM calmodulin (thin dashed line); 0.12 �g/ml phospha-tidylinositol (thick solid line); or 0.12 �g/ml phosphatidylinositol, 10 �M Ca2�,and 200 nM calmodulin (thick dashed line). The excitation wavelength was 387nM. The fluorescence intensities were normalized to the maximum emissionof the BFP fluorophore.

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in all BFP-PMCA-GFP molecules and it assumes that bothprobes can undergo unrestricted motion.Because of the lack of homology, the extreme N- and C-ter-

minal ends of the PMCA cannot be modeled with the aid of thecrystal structure of SERCA.On the basis of our results obtainedwith the BFP-PMCA-GFP protein, the ends of the PMCAwould be close to each other. This finding may explain theresults of domain switching showing that the autoinhibitorydomain C can be effective when placed at an N-terminal loca-tion (41).In contrast with the remarkable differences between the

E1Ca and E2(TG) conformers seen in SERCA crystals (3, 4), theintensity of FRET in BFP-PMCA-GFP did not exhibited a sig-nificant change upon the addition of Ca2�. The absence ofeffect of Ca2� may indicate that the relative position of thefluorophores in BFP-PMCA-GFP does not significantly changeduring the transition of E2 to E1 despite the rearrangement ofother domains in the protein. On the other hand, the intensityof FRET decreased when Ca2�-calmodulin was added indicat-ing that a modification in the relative position of the fluoro-phores took place during the activation of the PMCA. Thiseffect was partially reversed when Ca2� was removed and cal-modulin dissociated from the enzyme. The similar dependenceof FRET and the BFP-PMCA-GFP activation with the concen-tration of calmodulin reinforce the idea that the conforma-tional changemonitored by FRETwas specifically related to theactivation of the enzyme. Considering the substantial change inPMCA function upon activation, the estimated distance changebetween the fluorophores seems rather small. However, toachieve the optimal catalytic activity, different domains of thePMCAmay engage in conformational changes that do not nec-essarily involve a large change in the distance between the N-and C-terminal ends. Also because the change is an average ofall the labeled molecules, the estimated distance change uponactivation could be lowered by the presence of some moleculesthat were not activated. Furthermore, the changes observed in

the purified solubilized protein should be extrapolated withcaution to the PMCA in the natural membrane environment.It is well known that the PMCA can be activated by acidic

lipids. It has been proposed that the activation by acidic lipids isin part due to their binding to the calmodulin-binding sitemimicking the effects of calmodulin (38, 39). Consistently withthis idea phosphatidylinositol-activated BFP-PMCA-GFPshowed low FRET similar to that attained by the enzyme acti-vated by Ca2�-calmodulin in the presence of PC. This resultsuggests that the activation by acidic lipids, like that producedby calmodulin, involves a change in the relative position of theN- and C-terminal end of the protein.Although the BFP andGFP proteins are relatively resistant to

proteolytic attack, the PMCA is highly sensitive to cleavage atthe N- and C-terminal regions. The decrease of the BFP-PMCA-GFP FRETwith the extent of proteolysis was consistentwith the liberation of the fluorescent proteins from the PMCA,and it pointed out the essential role of the PMCA in bringingthe two fluorescent proteins together. We found that thedecrease of FRET with the time of proteolysis was biphasic.�25% of the total FRET intensity decayed rapidly concomi-tantly with the removal of the GFP from the BFP-PMCA-GFPwhile the remainder 75% decayed with a time course compati-ble with the removal of the N-terminal region containing theBFP. The persistence of FRET after the removal of GFP fromBFP-PMCA-GFP may reflect the interaction of a proteolyticfragment of BFP-PMCA and the cleaved C-terminal part of thepump containing GFP. In fact the C28W peptide resemblingthe calmodulin-binding autoinhibitory domain is capable ofinhibiting the pump activated by proteolysis but with less effi-ciency than the complete C-terminal autoinhibitory sequencewhen it is part of the PMCA molecule (42).The precise structural rearrangements of the PMCA mole-

cule involved in the activation process are not known. Recently,amodel for the activation of the PMCAby calmodulin has beenrecently proposed (43). According to this model the preferredroute leading to activation involves (i) the binding of calmodu-lin to domain C of an inhibited conformation of the pump,which has the C domain interacting with the catalytic region;(ii) a conformational change in the autoinhibitor promoted bythe binding of calmodulin and the detachment of the autoin-hibitor from the catalytic site; and (iii) a further rearrangementof the open conformation, which stabilizes the pump in an acti-vated state (Scheme 1).We have previously shown that the mutation D170N acti-

vates the pump and renders it almost insensitive to further acti-vation by calmodulin (19). Here, we found that BFP-PMCA(D170N)-GFP exhibited less FRET than the wild type in

2

1CaM

PMCA(c)

CaM. PMCA(c) CaM. PMCA(o)

CaM.PMCA(o) *

3

SCHEME 1

FIGURE 8. Emission spectra of BFP-PMCA(D170N)-GFP. The concentrationof BFP-PMCA(D170N)-GFP was adjusted at 26 nM in a standard media asdescribed under “Materials and Methods.” The sample was excited at a wave-length of 387 nm, and the fluorescence emission spectra were recorded (solidline). The spectra were taken again after the sequential addition of enoughCaCl2 to give 10 �M free Ca2� (long dashed line), 200 nM calmodulin (shortdashed line), and excess of 500 �M EGTA (dotted line). The fluorescence inten-sities were normalized to the maximum emission of the BFP fluorophore.

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agreement with the high activity of D170N mutant of thePMCA in the absence of calmodulin.The investigation of FRET in BFP-PMCA(D170N)-GFP

allowed a better characterization of the relationship betweenthe conformational change reported by FRET and the activa-tion of the PMCA. Because FRET in BFP-PMCA(D170N)-GFPwas not changed by calmodulin, the decrease in FRET is notlikely to be caused by the conformational changes producedduring the activation step 1 (Scheme 1). We have previouslyfound that, despite the high activity of mutant D170N, the rateof proteolysis of its C-terminal segment was still significantlyaccelerated by calmodulin, suggesting that themutation did notdisrupt the interaction between the autoinhibitor and its accep-tor site (19). Because the BFP-PMCA(D170N)-GFP FRET wasinsensitive to calmodulin the observed changes in FRETdid notseem to be related to the opening of the pump (step 2, Scheme1). Following this line of reasoning we propose that the changesin FRET in BFP-PMCA-GFP were caused by the interactionbetween the N and C segments during the final activated stateof the PMCA (step 3). The results obtained with BFP-PMCA(D170N)-GFP suggest that the change in the relativelocation of the ends of the PMCA, while occurring during acti-vation, may take place independently of the major autoinhibi-tory region. This finding is in line with our previous proposalthat the activated state may be attained without a full disen-gagement of the autoinhibitory domains from the catalyticcore. The finding that the relative location of the ends of thePMCA molecule are sensitive to the activation state of theenzyme seems surprising given the fact that neither the aminoacids segment near the N nor the C terminus has been reportedto have a functional role (8, 9, 32).Constructions of the type of BFP-PMCA-GFP characterized

here seem a promising tool for more detailed studies of theactivation process of PMCA. Furthermore, this strategy mayallow analysis of PMCAactivation in its specific environment inlive cells by FRET microscopy. In particular its application toother PMCA isoforms may help to clarify recent doubts as tothe actual importance in vivo of the differences in their calmod-ulin regulation deduced from in vitro assays (44).

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Activation-sensitive FRET in BFP-PMCA-GFP

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Gerardo R. Corradi and Hugo P. Adamo Pump Involved in the Activation2+Segments of the Plasma Membrane Ca

Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Intramolecular Fluorescence Resonance Energy Transfer between Fused

doi: 10.1074/jbc.M703377200 originally published online September 27, 20072007, 282:35440-35448.J. Biol. Chem. 

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